Discovery of Alternative Artemisinin Binding Sites in Plasmodium falciparum ATPase-6

Malaria is a fatal yet preventable and treatable disease. It is commonly spread through the bite of an infected Anopheles mosquito. Malaria parasites belong to the Plasmodium genus and can be caused by the falciparum, malariae, ovale, vivax, and knowlesi species. Artemisinin is an endoperoxide lactone extracted from qinghaosu (Artemisia annua L. or sweet wormwood). It and its derivatives possess uncharacteristically rapid action against Plasmodium falciparum. Artemisinin is unique in its effectiveness in deadly cerebral malaria. The endoperoxide bridge is crucial for its antimalarial activity; however, how it aids in killing the parasite is unknown. While there are multiple suggested modes of action (MOA) for artemisinin, none to date have a well-characterized protein target except for PfATP6, proposed by Krishna, et al. His work suggested that artemisinin binds to the homologous thapsigargin binding site of PfATP6. Herein, knowledge of the previously proposed mechanisms was utilized along with numerous computational techniques to determine a more detailed and plausible MOA for artemisinin against PfATP6. This led to the discovery of two new, putative PfATP6 binding sites for artemisinin. None of the previous work has explained the co-dependence of antimalarial efficacy on the concentration of Fe(II). In our work, we searched for putative sites containing a cysteine residue, Fe(II) and artemisinin in a conformation allowing for the well-accepted ring-opened C4 primary artemisinin carbon radical to form a covalent bond with a cysteine thiol rather than undergoing an intramolecular self-emolative diradical ring closure. Clearly there are geometric constraints for a transition state that side-steps the latter reaction and instead allows for interception by a cysteine thiol. We have suggested mechanistic possibilities for this capture by the artemisinin C4 radical and propose that PfATP6 is deactivated by blocking the Ca(II) channel by the modification of a cysteine at either C1031 or C92. It is alternatively possible that these modifications lead to alterations in the function of the protein, rendering it dysfunctional. Structure-based virtual screening was then used to screen a commercial database of compounds to find novel inhibitors of PfATP6. Biological testing will be done to determine if targeting these new sites can produce potent antimalarials with less structural complexity than artemisinin itself